Assay for inhibitors of CIP/KIP protein degradation

Abstract

An assay and system compatible with high throughput screening (HTS) that is capable of identifying inhibitors, such as small-molecule inhibitors, of the degradation of the Cdk inhibitor p21, are described. The assay is based on the use of fusion protein comprising (i) a p2 polypeptide; and (i) a reporter protein linked to the C-terminal of said p21 polypeptide, wherein the fusion protein has a half-life that is similar to that of the p21 polypeptide. Inhibitors identified by this assay may be useful to inhibit the proliferation of tumor cells, and thus for the treatment of cancers.

What is claimed is: 
     
       1. A method for determining whether a test compound inhibits the degradation of a Cip/Kip protein by the Ubiquitin-Proteasome system and may be useful for treating cancer, said method comprising
 (a) contacting said test compound with a cell expressing a fusion protein in the presence of an eukaryotic protein synthesis inhibitor, said fusion protein comprising (i) a Cip/Kip polypeptide; and (ii) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide, and 
 (b) measuring a readout signal from the reporter protein, 
 
       wherein a higher readout signal from the reporter protein in the presence of said test compound, relative to the readout signal in the absence of said test compound, is indicative that said test compound inhibits the degradation of a Cip/Kip protein by the Ubiquitin-Proteasome system and may be useful for treating cancer. 
     
     
       2. The method of  claim 1 , wherein the Cip/Kip polypeptide is a p21 polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a functional variant or fragment thereof having p21 activity. 
     
     
       3. The method of  claim 2 , wherein said half-life is from 30 minutes to about 1 hour. 
     
     
       4. The method of  claim 1 , wherein said protein synthesis inhibitor is cycloheximide (CHX). 
     
     
       5. The method of  claim 1 , wherein said reporter protein is a luciferase. 
     
     
       6. The method of  claim 5 , wherein said luciferase is a  Renilla  luciferase polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a functional variant or fragment thereof having  Renilla  luciferase activity. 
     
     
       7. The method of  claim 5 , wherein said readout signal from the reporter protein is bioluminescence in the presence of a luciferase substrate. 
     
     
       8. The method of  claim 7 , wherein said luciferase substrate is coelenterazine or an analog thereof. 
     
     
       9. The method of  claim 1 , wherein said cell further comprises an inducible expression system for inducible expression of the fusion protein. 
     
     
       10. The method of  claim 9 , wherein said inducible expression system is a tetracycline-controlled expression system. 
     
     
       11. A system for determining whether a test compound inhibits the degradation of a Cip/Kip protein by the Ubiquitin-Proteasomes system and may be useful for treating cancer, said system comprising:
 a cell expressing a fusion protein, said fusion protein comprising (i) a Cip/Kip polypeptide; and (ii) a reporter protein linked to the C-terminal of said Cip/Kip polypeptide, wherein said fusion protein has a half-life that is similar to that of said Cip/Kip polypeptide; 
 an eukaryotic protein synthesis inhibitor; and 
 a detection system to measure a readout signal from the reporter protein. 
 
     
     
       12. The system of  claim 11 , wherein the Cip/Kip polypeptide is a p21 polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a functional variant or fragment thereof having p21 activity. 
     
     
       13. The system of  claim 12 , wherein said half-life is from 30 minutes to about 1 hour. 
     
     
       14. The system of  claim 11 , wherein said protein synthesis inhibitor is cycloheximide (CHX). 
     
     
       15. The system of  claim 11 , wherein said reporter protein is a luciferase. 
     
     
       16. The system of  claim 15 , wherein said luciferase is a  Renilla  luciferase polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a functional variant or fragment thereof having  Renilla  luciferase activity. 
     
     
       17. The system of  claim 15 , wherein said system further comprises a luciferase substrate. 
     
     
       18. The system of  claim 17 , wherein said luciferase substrate is coelenterazine or an analog thereof. 
     
     
       19. The system of  claim 11 , wherein said cell further comprises an inducible expression system for inducible expression of the fusion protein. 
     
     
       20. The system of  claim 19 , wherein said inducible expression system is a tetracycline-controlled expression system.